Method of sustaining aroma and use thereof

ABSTRACT

The first object of the present invention is to provide a method for preparing an aroma-retaining material, which enables to increase the adsorbed aroma amount, retain the aroma for a long period of time, and impart stability. The second object of the present invention is to provide an aroma-retaining material, which can retain aroma for a long period of time and has satisfactory stability. The third object of the present invention is to provide a composition comprising the aroma-retaining material. The forth object of the present invention is to provide an aroma-retaining agent, which enables to stabilize an aromatic substance. The fifth object of the present invention is to provide a retained-releasing bactericide comprising the aroma-retaining material. The present invention solves the above objects by providing a method for retaining an aroma in such a manner of mixing an aromatic substance and a cyclic tetrasaccharide or the mixture of cyclic tetrasaccharide and its saccharide derivative(s), an aroma-retaining material obtainable by the method, a composition comprising the aroma-retaining material, an aroma-retaining agent comprising a cyclic tetrasaccharide or a mixture of a cyclic tetrasaccharide and its saccharide derivative(s) as effective ingredients, and a bactericide using the aroma-retaining material having a sustained-release property.

TECHNICAL FIELD

The present invention relates to a method for retaining aroma and usethereof, more particularly, to a method for retaining aroma,aroma-retaining materials obtainable by the method; compositionscomprising the aroma-retaining materials, such as foods, cosmetics,pharmaceuticals and commodities; aroma-retaining agents; andbacteriostats and/or bactericides comprising the aroma-retaining agentshaving a sustained-release property.

BACKGROUND ART

The following methods have been known as methods for retaining aroma;those comprising a step of (i) allowing an aromatic substance to adsorbon an involatile substance; (ii) covering an aromatic substance with amembrane made of an impermeable material; (iii) preventing the diffusionof an aromatic substance by lowering the moisture content of a product;or (iv) allowing an aromatic substance to form an inclusion complex withother substances, reported by Sugisawa, H., “Kagaku to Seibutsu”,Vol.10, No.2, p.92, 1972).

The use of various sacchaides for retaining aroma has been proposed. Forexample, Japanese Patent Kokoku No. 37,062/77 discloses a method ofadmixing an aromatic substance with an oligosaccharide to form a solidmaterial; Japanese Patent Kokoku No. 52,177/93 discloses the method ofadmixing an oily aromatic substance with anhydrous crystallineα-maltose, which was proposed by the same applicant as the presentinvention; Japanese Patent Kokoku No. 26,345/96 discloses a mothod ofadmixing an oily aromatic substance with anhydrous crystalline α-maltoseand cyclodextrin to prepare a solid mixture, which was proposed by thesame applicant; Japanese Patent Kokai No. 35,251/79 and Kokoku No.986/93 disclose a method of allowing an aromatic substance to forming aninclusion complex with cyclodextrin. Due to the saccharides used, thesemethods, however, have some disadvantages of lesser adsorption level ofaromatic substances, insufficient capability of retaining aromaticsubstances, restricted applicability to specific oily aromaticsubstances, and unsatisfactory solubility of the resulting mixture intheir secondary proceedings.

α,α-Trehalose, whose industrial production has been developed in recentyears, is useful for retaining aroma. For example, Japanese Patent KokaiNo. 111,284/97 discloses a method for producing emulsified perfumeries,comprising the steps of admixing an aromatic substance withα,α-trehalose in the presence of water and an emulsifier and powderyaroma-retaining materials made from the emulsified perfumeries, anddrying the mixture. Although these trehalose products have a relativelyhigh aroma retaining property and stability, they have disadvantages onthe adsorbed amount of aroma and the capability of retaining aroma dueto their easy crystallization. Therefore, there still remains anecessity of finding more suitable saccharides.

Recently, a cyclic tetrasaccharide represented by Chemical Formula 1(hereinafter, it is simply abbreviated as “CTS”) was reported byBradburk G. M., et al., Carbohydrate Research, Vol. 329, pp. 655-665,(2000). CTS is a non-reducing cyclic saccharide composed of four glucosemolecules linked via the alternating α-1,3 and α-1,6 glycosidic bonds.The process disclosed therein was not suitable as an industrial scaleproduction, and it was not revealed on its use and physical property indetail. As disclosed in International Publication Nos. WO 90,338/01(PCT/JP01/04276) and WO 10,361/02 (PCT/JP01/06412), the presentinventors have established the process for producing CTS from starch andphytoglycogen with a low cost. Chemical Formula 1:

DISCLOSURE OF INVENTION

The object of the present invention is to solve the above problems ofconventional methods for preparing aroma-retaining materials.Concretely, usual aromatic substances tend to denature, deteriorate andevaporate even when stored for a relatively short period of time due tolesser adsorption level of aromatic substance, insufficient capabilityof retaining aroma, and insufficient stability of aroma-retainingmaterials. The first object of the present invention is to provide amethod for retaining aroma useful for various fields without fear ofaffecting aroma and aroma-retaining materials for the purpose ofincreasing the retaining amount of aroma, the long term reservation ofaroma contained in aroma-retaining materials, and imparting stability toaroma-retaining materials. The second object of the present invention isto provide an aroma-retaining material having a satisfactory stabilityand capability of retaining aroma for a relatively long period of time.The third object of the present invention is to provide a compositioncomprising an aroma-retaining material. The fourth object of the presentinvention is to provide an aroma-retaining agent capable of retainingaroma for a relatively long period of time. The fifth object of thepresent invention is to provide a bacteriostat and/or bactericidecomprising the agent having a sustained-release property.

The present inventors have eagerly studied the use of some kinds ofsaccharides, particularly CTS (industrially produced form starch orphytoglycogen material) or a mixture of CTS and its saccharidederivative(s) to attain the above objects. As a result, they found thatCTS or a mixture of CTS and its saccharide derivative(s) was useful forretaining aroma of an aromatic substance. They accomplished the presentinvention by establishing a novel method for retaining aroma, a novelaroma-retaining material, a novel composition comprising anaroma-retaining material, a novel agent containing CTS or a mixture ofCTS and its saccharide derivative(s), and a novel bacteriostat and/orbactericide comprising the agent having a sustained-release property.

BEST MODE FOR CARRYING OUT THE INVENTION

Aromatic substance as referred to as in the present invention is notrestricted to a specific one, for example; extracts from citrus such asan orange, lemon, lime, and grapefruit; vegetable oils such as anessential flower oil, peppermint oil, spearmint oil, spice oil, and herboil; plant extracts such as a kola nut extract, coffee extract, vanillaextract, cocoa extract, black tea extract, spice extract, and herbextract; essential oils extracted from an animal tissue; natural aromamaterials including a tissue containing thereof; derivatives of naturalaroma materials, chemically or semichemically synthesized aromaticsubstances, and mixtures thereof; can be used in the present invention.Further, compositions comprising aromatic substances for seasoning othercompositions or fermented foods can be used in the present invention,for example; alcohols such as “sake” (Japanese rice wine), whiskey,brandy, liqueur, and mirin (Japanese sweet rice wine); dried bonitoflake; boiled-dried fish; dried squid; oarweed; shrimp; shiitakemushroom; or extracts thereof; extracts such as bouillon, fisheryextracts, vegetable extracts, and fruit extracts, or seasonings from theextracts; fermented foods such as soy sauce, fish sauce, miso (soybeanpaste), mirin, vinegar, and alcohols.

CTS or a mixture of CTS and its saccharide derivative(s) usable foradmixing with aromatic substances or compositions comprising aromaticsubstances is not specifically limited on their origin and preparation.For example, they can be produced in a usual fermentation, enzyme, ororganic-chemistry technique. The reaction mixtures prepared by the abovemethods can be used intact as CTS or a mixture of CTS and its saccharidederivative(s). Optionally, it can be partially or perfectly purifiedbefore use. CTS or a mixture of CTS and its saccharide derivative(s) canbe enzymatically produced from amylaceous materials or saccharidederivatives thereof with as following; (i) converting panose into CTS byan α-isomaltosyl transferring enzyme, which was disclosed by the presentinventors in International Publication No. WO 90,338/01; (ii) directlyproducing CTS or a mixture of CTS and its saccharide derivative(s) fromamylaceous substances using α-isomaltosyl glucosaccharide-forming enzymeand α-isomaltosyl transferring enzyme in combination, which wasdisclosed by the same applicant as the present invention inInternational Publication No. WO 10,361/02. Since these enzymaticmethods produce CTS or its saccharide derivative(s) from abundant andinexpensive amylaceous substances efficiently and at a low cost, theyare useful for industrial production of them. CTS has been known to bein various forms such as amorphous anhydrate, crystalline anhydrate,crystalline monohydrate and crystalline pentahydrate. CTS used in thepresent invention can be any of such forms. Particularly, when anaroma-retaining material in the form of a powder or solid is produced bymixing CTS and an aromatic substance containing moisture, CTS in theform of a crystalline anhydrate, crystalline monohydrate or amorphousanhydrate can be advantageously used because they act as a dehydratingagent having an advantageous of dehydrating capability.

Materials comprising other sacccharides alone with CTS and itssaccharide derivative(s) can be also used in the present invention.“Saccharide derivatives of CTS” as referred to as in the presentinvention means derivatives of CTS having one or more glycosyl groupswhich are same or different each other. A mixture of CTS and itssaccharide derivative(s) is usually in the form of a saccharide solutioncontaining CTS, its saccharide derivative(s) (having one or moreglucoses positioning at one or more hydroxly groups in CTS), and/orother saccharides such as glucose, maltooligosaccharides, andmaltodextrins obtainable by allowing α-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyl-transfer enzyme incombination to act on amylaceous substances. Further, it can be purifiedusing ion-exchange resins partially or perfectly. According to JapanesePatent Application No. 67,282/01, disclosed by the same inventors as thepresent invention, saccharide derivatives which are formed bytransferring one or more glucose residues (e.g. α-D-glucopyranosyl,β-D-galactopyranosyl and β-D-chitosaminyl) to one or more hydroxylgroups of CTS or its saccharide derivative(s), and which are produced byallowing one or more saccharide transferring enzymes (e.g.cyclomaltodextrin glucanotransferase, β-galactosydase, α-galactosydase,and lysozyme) to act on the mixture in the presence of saccharidesubstrates (e.g. monosaccharide, oligosaccharide, and polysaccharide)can be also used. Furthermore, the above saccharide derivatives can bepurified partially or perfectly.

The aroma-retaining material of the present invention can be prepared byadmixing (i) an aromatic substance(s) or a composition thereof, (ii) CTSor a mixture of CTS and its saccharide derivative(s) in the form of asyrup, mascuite, solid, or powder, (iii) optional water, and (iv)optional other ingredients. When the resulting aroma-retaining materialis in the form of a liquid or semi-solid, it can be converted into theform of a solid or powder by conventional drying such as heating, dryingin vacuo, spray-drying, or lyophilizing. Particularly, thearoma-retaining material in a powder form can be advantageously producedby the steps of homogeneously admixing aromatic substances andsaccharides with water and/or emulsifier, and drying the resultingmixture.

CTS or a mixture of CTS and its saccharide derivative(s) used for mixingwith an aromatic substance can be freely chosen from those with variousforms such as syrup, mascuite, solid, powder, and mixture thereof,considering the property of the aromatic substance or other ingredients,stability of the aromatic substance, forms of aroma-retaining materials,and workability. The term “solid or powder containing CTS or a mixtureof CTS and its saccharide derivative(s)” as referred to as in thepresent invention means a saccharide mixture in the form of a solid orpowder, which contains (i) CTS amorphous anhydrate, CTS crystallineanhydrate, CTS crystalline hydrate, CTS crystalline anhydrate, or amixture thereof, (ii) one or more saccharide derivatives of CTS in theform or a solid or powder, and (iii) other saccharides in the form of asolid or powder.

The composition comprising aromatic substances with CTS or a mixture ofCTS and its saccharide derivative(s) is useful as an aroma-retainingagent. Further, it can be used in an appropriate amount of one or moremonosaccharides such as xylose, arabinose, glucose, fructose, psicose,and mannose, oligosaccharides such as maltose, isomaltose, sucrose,lactose, α,α-trehalose, neotrehalose, panose, maltotriose,maltotetraose, maltopentaose, α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, and derivatives thereof, polysaccharide such ashigh-molecular dextrin, guar gum, arabic gum, pullulan, and hydroxyethylstarch. Furthermore, intense sweeteners such as sucralose, acesulfame-K,stevia, glycotransferring stevia, glycyrrhizin, saccharine, andL-aspartyl-L-phenylalanine methylester can be freely used.

Aroma-retaining materials having retaining property and stability can beobtained by mixing the solid and/or powder containing CTS or a mixtureof CTS and its saccharide derivative(s) with an aromatic substance or acomposition containing aromatic substances, optionally dissolving themixture in an organic solvent, and, optionally, adding other ingredientsto the resulting mixture. By co-existing these saccharides and aromaticsubstance containing organic solvent in an amount sufficient to dissolvea part of the saccharides, and optionally, other ingredients, solidproducts can be obtained. Further, the resulting solid products can befreely processed into powdery aroma-retaining products.

The term “allowing CTS or a mixture of CTS and its saccharidederivative(s), aroma-retaining materials, and bacteriostat orbacteriocide to incorporate into” as referred to as in the presentinvention means contacting the above ingredients with other ingredientsaccording to usual methods of mixing, kneading, dissolving, melting,dispersing, suspending, emulsifying, soaking, permeating, dispersing,applying, coating, spraying, injecting, crystallizing, and solidifying.The term “allowing to co-exist with aroma-retaining materials in theform of a bacteriostat or bacteriocide” as referred to as in the presentinvention means separately co-existing an aroma-retaining material(s)and a product as in the case of using the function of a vaporizingaromatic substance, for example, co-existing the aroma-retainingmaterials placed in a sealed or semi-sealed container.

Proportion of CTS or a mixture of CTS and its saccharide derivative(s)to an aromatic substance is not specifically restricted as long as thearomatic substance is satisfactorily adsorbed and retained, usually, inan amount of 1 to 10,000, desirably, 10 to 5,000 parts by weight to onepart by weight of an aromatic substance(s) on a dry solid basis. Whenthe amount of CTS or the mixture is lower than the lower limit, theproperty of retaining aroma will be more insufficient although theamount of the adsorbed aroma substance by weight is larger. While, whenthe amount of CTS or the mixture is higher than the upper limit, theworkability during preparation and the physical properties of thecomposition will become worse.

The aroma-retaining material of the present invention is suitable as anagent for retaining aroma and/or sustained-releasing agent containingaromatic substances because it inhibits or prevents aromatic substancesto evaporate and disappear, and prolongs the time until completion ofits evaporating and disappearing, and retaining the aroma for a relativelong period of time. Examples of such are alcohols, seasonings includingsoup stocks and fermented foods can be preserved for a relatively longperiod of time while retaining their inherent flavors of aromaticsubstances and keeping their original quality. Aromatic substanceshaving an additional effect can be used as a preparation graduallyperforming the effect. Compositions, having the effect as a bacteriostatand/or bactericide or repellent, such as alcohols including ethanol,organic acids including acetic acid, hinokithiol, and wasabiol, can beadvantageously used for products susceptive to the affect ofmicroorganic contaminants or harmful insects by mixing or co-existingthe aroma-retaining materials in foods, cosmetics, pharmaceuticals,clothing, or commodities because they gradually released aromaticsubstances and exert effect of bacterostat and/or bacterocide orrepellent. Compositions having some effects on mind and body and/or theprevention and treatment of diseases, such as essential oils andextracts form herbs including lavender, rosemary, and chamomile, can befreely used as a deodorizer gradually releasing aromatic substances or asource of aromatic substances for aroma therapy such as a bath or herbtea by dissolving into a hot water.

The aroma-retaining material obtainable by the above-mentioned methodscan be used as the following food compositions; beverages; powderedbeverages; seasonings; sweeteners; Japanese confectioneries; cakes;water ices; syrups; pastes; pickles; processed marine products;processed livestock food products; prepared foods; dairy products;retort pouches; health foods; feeding stuffs and pet foods.Particularly, the aroma-retaining material of the present invention issuitable as a raw material for health foods because it retains aroma fora relatively long period of time and exerts the effect for intestinaldisorders and decreases cholesterol due to the effect as a dietary fiberby the contained CTS or the mixture of CTS and its saccharidederivative(s). Furthermore, the aroma-retaining material can be used ascosmetics such as a soap, shampoo, rinse, body lotion, tooth paste, lipcream, agent for hair restoration, hairdressing, and bath salt,pharmaceuticals such as pack, buccal, cataplasm, oral agent, agent forpercutaneous absorption, agent for permucotaneous absorption, powderyagents, and tablet, commodities such as agent for bed bath, detergent,fabric softener, fabric conditioner, aromatic, deodorant, reodorant,perfume, bacteriocide, fungicide, and repellent.

Varying depending on the kind and the form of a final product such as afood, beverage, pharmaceutical, and commodity, usually it contains 0.001to 50 parts by weight of the aroma-retaining material to the rawmaterials or final products.

Following examples explain the present invention in more detail, but thepresent invention must not be restricted by the examples.

First, examples of producing enzymes for preparing CTS or a mixture ofCTS and its saccharide derivative(s) are explained as follows:

EXAMPLE A-1

Production of Enzymes for Producing CTS and its Saccharide Derivatives

α-Isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme required for producing CTS from starchmaterial were prepared at first. A liquid culture medium, consisting of4.0% (w/v) of “PIN-DEX #4”, a partial starch hydrolyzate commercializedby Matsutani Chemical Industries., Co. Ltd., Tokyo, Japan, 1.8% (w/v) of“ASAHIMEAST”, a yeast extract commercialized by Asahi Breweries, Ltd.,Tokyo, Japan, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodiumphosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, andwater, was placed in 500-ml Erlenmeyer flasks in a volume of 100 mleach, sterilized by autoclaving at 121° C. for 20 min, cooled, and thenseeded with Bacillus globisporus C9 strain, FERM BP-7143, followed byculturing under rotary-shaking conditions at 27° C. and 230 rpm for 48hours for seed culture. About 20 L of a fresh preparation of the sameliquid culture medium as used in the above seed culture were placed in a30-L fermentor, sterilized by heating, and then cooled to 27° C. andinoculated with 1% (v/v) of the seed culture, followed by culturing at27° C. and pH 6.0-8.0 for 48 hours under aeration-agitation conditions.After completion of the culture, the resulting culture, which had about0.45 unit/ml of α-isomaltosylglucosaccharide-forming enzyme, about 1.5units/ml of α-isomaltosyl-transferring enzyme, and about 0.95 unit/ml ofcyclic tetrasaccharide-forming activity, was centrifuged at 10,000 rpmfor 30 min to obtain about 18 L of a supernatant. When measured forenzymatic activity, the supernatant had about 0.45 unit/ml of theα-isomaltosylglucosaccharide-forming enzyme, i.e., a total enzymaticactivity of about 8,110 units; about 1.5 units/ml ofα-isomaltosyl-transferring enzyme, i.e., a total enzymatic activity ofabout 26,900 units.

The activities of these enzymes were measured as follows: The activityof α-isomaltosylglucosaccharide-forming enzyme was measured by the stepsof dissolving maltotriose in 100 mM acetate buffer (pH6.0) to give aconcentration of 2% (w/v) for a substrate solution, adding a 0.5 ml ofan enzyme solution to a 0.5 ml of the substrate solution, enzymaticallyreacting the mixture solution at 35° C. for 60 min, suspending thereaction mixture by boiling for 10 min, and quantifying maltose, amongthe isomaltosyl maltose and maltose formed in the reaction mixture, onconventional HPLC. One unit activity of theα-isomaltosylglucosaccharide-forming enzyme is defined as the amount ofthe enzyme that forms 1 μmole of maltose per minute under the aboveconditions. Throughout the specification, the activity of theα-isomaltosylglucosaccharide-forming enzyme means the unit(s) measuredas above.

The activity of α-isomaltosyl-transferring enzyme was measured by thesteps of dissolving panose in 100 mM acetate buffer (pH6.0) to give aconcentration of 2% (w/v) for a substrate solution, adding a 0.5 ml ofan enzyme solution to 0.5 ml of the substrate solution, enzymaticallyreacting the mixture by boiling for 10 min, and quantifying glucose,among the CTS and glucose formed in the reaction mixture, by the glucoseoxidase method. One unit activity of the α-isomaltosyl-transferringenzyme is defined as the enzyme amount that forms 1 μmole of glucose perminute under the above enzymatic reaction conditions. Throughout thespecification, the enzymatic activity of the α-isomaltosyl-transferringenzyme means the unit(s) measured as above.

The cyclic tetrasaccharide-forming activity is measured by the steps ofdissolving “PINE-DEX #100”, a partial starch hydrolysate commercializedby Matsutani Chemical Industries., Co., Ltd., Tokyo, Japan, in 50 mMacetate buffer (pH 6.0) to give a concentration of 2% (w/v) for asubstrate solution, adding 0.5 ml of an enzyme solution to 0.5 ml of thesubstrate solution, enzymatically reacting the mixture solution at 35°C. for 60 min, suspending the reaction mixture by boiling for 10 min,and then further adding to the resulting mixture one milliliter of 50 mMacetate buffer (pH 5.0) with 70 units/ml of “TRANSGLUCOSIDASE L AMANO™”,an α-glucosidase commercialized by Amano Pharmaceutical Co., Ltd.,Aichi, Japan, and 27 units/ml of glucoamyrase, commercialized by NagaseBiochemicals, Ltd., Kyoto, Japan, and incubated at 50° C. for 60 min,inactivating the retaining enzymes by heating at 100° C. for 10 min, andquantifying cyclotetrasaccharide on HPLC similarly as in Experiment 1.One unit of cyclotetrasaccharide-forming activity is defined as theenzyme amount that forms one micromole of cyclotetrasaccharid per minuteunder the above enzymatic reaction conditions. Throughout thespecification, the cyclic tetrasaccharide-forming activity means theactivity (units) measured as above.

EXAMPLE A-2

Preparation of Enzymes Derived Form Bacillus globisporus C9

About 18 L of the supernatant in Example A-1 was salted out with 80%saturated ammonium sulfate and allowed to stand at 4° C. for 24 hours,and the formed sediments were collected by centrifugation at 10,000 rpmfor 30 min, dissolved in 10 mM phospate buffer (pH7.5), and dialyzedagainst a fresh preparation of the same buffer to obtain about 400 ml ofa crude enzyme solution with 8,110 units of theα-isomaltosylglucosaccharide-forming enzyme, 24,700 units ofα-isomaltosyl-transferring enzyme, and about 15,600 units of cyclictetrasaccharide-forming activity. The crude enzyme solution wassubjected to ion-exchange chromatography using 1,000 ml of “SEPABEADSFP-DA13” gel, an ion-exchange resin commercialized by MitsubishiChemical Industries, Ltd., Tokyo, Japan. Theα-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferring enzyme were eluted as non-adsorbed fractions withoutadsorbing on the ion-exchange resin. The resulting enzyme solution wasdialyzed against 10 mM phosphate buffer (ph7.0) with 1 M ammoniumsulfate, and the dialyzed solution was free from impurities bycentrifuging, and subjected to affinity chromatography using 500 ml of“SEPHACRYL HR S-200”, a gel commercialized by Amersham Corp., Div.Amersham International, Arlington Heights, Ill., USA. Enzymaticallyactive components were adsorbed on the gel, and when sequentially elutedwith a linear gradient decreasing from 1 M to 0 M of ammonium sulfateand a linear gradient increasing from 0 mM to 100 mM of maltotetraose,the α-isomaltosylglucosaccharide-forming enzyme and theα-isomaltosyl-transferring enzyme were separatory eluted, i.e., theformer was eluted with the linear gradient of maltotetraose at about 30mM and the latter was eluted with the linear gradient of ammoniumsulfate at about 0 M. Thus, fractions with α-isomaltosyl-transferringactivity and those with the α-isomaltosylglucosaccharide-formingactivity.

Methods for separatory purifying theα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme are described in the below:

EXAMPLE A-3

Purification of α-isomaltosylglucosaccharide-Forming enzyme Derived fromBacillus globisporus C9

A fraction of the α-isomaltosylglucosaccharide-forming enzyme, obtainedexample A-2, was dialyzed against 10 mM phosphate buffer (pH 7.0)containing 1 M ammonium sulfate. The dialyzed solution was free frominsoluble impurities by centrifuging and fed to hydrophobicchromatography using 350 ml of “BUTYL-TOYOPEARL 650M”, a gelcommercialized by Tosoh Corporation, Tokyo, Japan. The enzyme wasadsorbed on the gel and eluted at about 0.3 M ammonium sulfate wheneluted with a linear gradient decreasing from 1 M to 0 M of ammoniumsulfate, followed by collecting fractions with the enzyme activity. Thefractions were pooled and again dialyzed against 10 mM phosphate buffer(pH 7.0) containing 1 M ammonium sulfate. The resulting dialyzedsolution was centrifuged, and the resulting supernatant free frominsoluble impurities was fed to affinity chromatography using “SEPHACRYLHR S-200” gel to purify the enzyme.

EXAMPLE A-4

Purification of α-isomaltosyl-Transferring Enzyme Derived Form Bacillusglobisporus C9

The fraction of the α-isomaltosyl-transferring enzyme, separated fromthe α-isomaltosylglucosaccharide-forming enzyme using the affinitychromatography described in Example A-2, was dialyzed against 10 mMphosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The dialyzedsolution was centrifuged, and the resulting supernatant free frominsoluble impurities was fed to hydrophobic chromatography using 350 mlof “BUTYL-TOYOPEARL 650M”, a gel commercialized by Tosoh Corporation,Tokyo, Japan. The enzyme was adsorbed on the gel and eluted at about 0.3M ammonium sulfate when eluted with a linear gradient decreasing from 1M to 0 M of ammonium sulfate, followed by collecting fractions with theenzyme activity. The fractions were pooled and again dialyzed against 10mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. Theresulting dialyzed solution was centrifuged, and the resultingsupernatant free from insoluble impurities was fed to affinitychromatography using “SEPHACRYL HR S-200” gel to purify the enzyme.

Following explains the method for preparing the mixture of CTS and itssaccharide derivative(s).

EXAMPLE B-1

Preparation of a Syrup Containing a Mixture of CTS and its SaccharideDerivative(s)

A potato starch was prepared into an about 6% starch suspension, admixedwith calcium chloride to give a final concentration of 0.1%, adjusted topH 6.0, further admixed with an α-amylase. To the resulting liquefiedsolution was added 2 units of α-isomaltosylglucosaccharide-formingenzyme prepared in Example A-3 and 6 units of α-isomaltosyl-transferringenzyme prepared in Example A-4 per gram starch on dry solid basisfollowed by the enzymatic reaction for 48 hours. The reaction mixturewas heated to and kept at 95° C. for 10 min to be inactivated, and then,it was decolored, desalted, filtered, and concentrated by conventionalmethods. As a result, an 80% saccharide syrup containing 0.6% glucose,1.5% isomaltose, 12.3% maltose, 63.5% CTS, and 5.2% saccharidederivatives of CTS combining with one or more glucoses was prepared.

EXAMPLE B-2

Preparation of CTS

About 100 L of a 4% (w/v) aqueous solution of corn phytoglycogen,commercialized by Q.P. Corporation, Tokyo, Japan, was prepared, adjustedto pH 6.0 and 30° C., and then admixed with 1 unit/g solid of a purifiedspecimen of α-isomaltosylglucosaccharide-forming enzyme obtained inExample A-3 and 10 units/g solid of a purified specimen ofα-isomaltosyl-transferring enzyme obtained in Example A-4, followed bythe incubation for 48 hours. After completion of the reaction, thereaction mixture was heated at 100° C. for 10 min with the aim ofinactivating the retaining enzymes. The resulting reaction mixture wasadjusted to pH 5.0 and 45° C., and then treated with α-glucosidase andglucoamylase similarly as in Example A-1 to hydrolyze the retainingreducing oligosaccharides, etc. The resulting mixture was adjusted to pH5.8 by the addition of sodium hydroxide and then incubated at 90° C. forone hour with the aim of inactivating the retaining enzymes and filteredwith the aim of removing insoluble substances. The filtrate wasconcentrated using a reverse osmosis membrane to give a concentration ofabout 16% on a dry solid basis, and the concentrate was in a usualmanner decolored, desalted, filtered, and concentrated. As a result,about 6.2 kg of a saccharide solution with a solid content of about3,700 g was obtained.

The saccharide solution as fed to a column packed with about 225 L of“AMBERLITE CR-1310 (Na-form)”, an ion-exchange resin commercialized byJapan Organo Co., Ltd., Tokyo, Japan, and chromatographed at a columntemperature of 60° C. and a flow rate of about 45 L/h. While thesaccharide composition of eluate from the column was monitoring by HPLCas described in Example A-1, fractions of CTS with a purity of at least98% were collected.

EXAMPLE B-3

Preparation of CTS Crystalline Hydrate

A fraction of CTS with a purity of at least 98%, obtained by the abovemethod, was concentrated by evaporation to give a concentration of about50% on a dry solid basis. About 5 kg of the concentrate was placed in acylindrical plastic vessel and then crystallized to obtain a whitecrystalline powder by lowering the temperature of the concentrate from65° C. to 20° C. over about 20 hours under gentle rotatory conditions.The above crystallized concentrate was separated by passing through acentrifugal filter to obtain 1,360 g of a crystalline product by wetweight, which as then further dried at 60° C. for three hours to obtain1,170 g of a crystalline powder of CTS. HPLC measurement of thecrystalline powder revealed that it contained CTS with a quite highpurity of at least 99.9%. When analyzed on powder X-ray diffractionanalysis, the CTS in a crystalline powder form had a diffractionspectrum having characteristic main diffraction angles (2θ) of 10.1°,15.2°, 20.3°, and 25.5°. The Karl Fischer method of the crystallinepowder revealed that it had a moisture content of 13.0%, resulting in afinding that it as a crystal of CTS having five moles of water per onemole of the crystal.

EXAMPLE B-4

Preparation of CTS Crystalline Monohydrate

CTS crystalline pentahydrate in the form of a powder, obtained accordingto the method in Example B-3, was placed in a glass vessel, and kept inan oil bath, which had been preheated at 140° C., for 30 min. The powderX-ray diffraction analysis of the CTS powder thus obtained gave acharacteristic diffraction spectrum having main diffraction angles (2θ)of 8.3°, 16.6°, 17.0°, and 18.2°. The Karl Fischer method of thecrystalline powder revealed that it had a moisture content of about2.7%, resulting in a finding that it was a crystal of CTS having onemole of water per one mole of the crystal.

EXAMPLE B-5

Preparation of CTS Crystalline Anhydrate

CTS crystalline pentahydrate in the form of a powder, obtained by themethod in Example B-3, was dried in vacuo at 120° C. for 16 hours. Thepowder X-ray diffraction analysis of the above CTS gave characteristicdiffraction spectra having main diffraction angles (2θ) of 10.8°, 14.7°,15.0°, 15.7°, and 21.5°. The Karl Fischer method of the resultingcrystalline powder revealed that it had a moisture content of about0.2%, meaning that it was substantially anhydrous.

EXAMPLE B-6

Preparation of CTS Amorphous Anhydrate

Fractions containing CTS with a purity of at least 98%, obtained by themethod in Example B-2, were in a usual manner desalted, decolored, andfiltered to obtain a concentrate having a solid concentration of 50%.The concentrate thus obtained was promptly freezed at −80° C.,lyophilized, and further dried in vacuo at 80° C. for three hours. Theresulting dried product was pulverized with a pulverizer. The powderX-ray diffraction analysis of the resulting powder revealed that thepowder was amorphous since it gave no characteristic diffractionspectrum. The Karl Fischer method of the powder revealed that it had amoisture content of about 0.3%, meaning that it was substantiallyanhydrous.

EXAMPLE C-1

Preparation of Aroma-Retaining Material Using CTS CrystallinePentahydrate, CTS Crystalline Monohydrate, CTS Crystalline Anhydrate, orCTS Amorphous Anhydrate

CTS crystalline pentahydrate, prepared by the method described inExample B-3, CTS crystalline monohydrate, prepared by the methoddescribed in Example B-4, CTS crystalline anhydrate, prepared by themethod described in Example B-5, or CTS amorphous anhydrate prepared bythe method described in Example B-6, was used for preparation ofaroma-retaining material. “ISOELEAT P™”, a branched cyclodextrincommercialized by Maruha corporation, Tokyo, Japan, anhydrous trehaloseprepared by the method described in Japanese Patent Kokai No.111,284/97, “PINEFIBRE™”, a dextrin commercialized by Matsutani ChemicalIndustries Co., Ltd., Tokyo, Japan, or soluble starch in a reagent gradecommercialized by Katayama Chemical Industries, Co., Ltd., Tokyo, Japan,was dried in vacuo at 80° C. for 16 hours as control saccharides.Ethanol or acetic acid in a liquid form as an aromatic substance wasgradually admixed with 10 g of the above saccharide, and the mixture wasplaced into a glass triturator and stirred with a glass rod until theresulting mixture did not keep a powder form. The resulting mixture wasweighed. The weight of ethanol or acetic acid was calculated bysubtracting the pre-measured saccharide weight. The weight of ethanol oracetic acid adsorbed on 1 g of each saccharide was in Table 1. TABLE 1Amount of Adsorbed Ethanol/Acetic Acid(g) Saccharide Ethanol Acetic acidCTS crystalline pentahydrate 0.32 0.27 CTS crystalline monohydrate 0.450.35 CTS crystalline anhydrate 0.50 0.47 CTS amorphous anhydrate 0.490.47 Branched cyclodextrin 0.30 0.23 Anhydrous trehalose 0.25 0.18Dextrin 0.21 0.26 Soluble starch 0.12 0.06

From the result in Table 1, CTS crystalline pentahydrate, CTScrystalline monohydrate, CTS crystalline anhydrate or CTS amorphousanhydrate has stronger effect on adsorbing ethanol or acetic acid in aliquid form than the control saccharides. Particularly, it is revealedthat CTS crystalline anhydrate and CTS amorphous anhydrate have thestrongest effect.

EXAMPLE C-2

Effect of Suppressing Volatilization

About 5 g of an ethanol-retaining material with CTS crystallineanhydrate, branched cyclodextrin, anhydrous trehalose, or dextrin,prepared freshly by the method described in Example C-1, was taken intoa 20 ml volume of glass vials. The vials were placed under normalpressure and room temperature for the purpose of evaporating ethanol.The amount of retained ethanol in samples freshly prepared, or after twohours later, four hours later, eight hours later, or 24 hours later, wasmeasured by dissolving each sample in about 10 ml of water, diluting itwith water to give a volume of 100 ml in a glass measuring flask, andmeasuring the amount of ethanol dissolved in the resulting aqueoussolution by a gas chromatography. In the case of a soluble starch as asample, additional centrifuging was carried out after diluting up to 100ml. The above gas chromatography was carried out using “GC-14B” (a gasliquid chromatography commercialized by Shimazu Corporation, Kyoto,Japan) equipped with “TC-5” (a capillary column sized 0.53 mm indiameter and 15 m in length, commercialized by GL Sciences Inc., Tokyo,Japan). The amount of retained ethanol per 1 g of each material is inTable 2. TABLE 2 Amount of Retained Ethanol (mg) Freshly- 2 h 4 h 8 h 24h Saccharide Prepared Later Later Later Later CTS crystalline 333 335290  10  5 anhydrate (100%)  (101%)  (87%) (3%) (2%) Branchedcyclodextrin 230 110 30 7 3 (100%) (48%)  (13%) (3%) (1%) Anhydroustrehalose 203 103 17 8 5 (100%) (51%) (8%) (4%) (2%) Dextrin 176  27  96 4 (100%) (15%) (5%) (3%) (2%) Soluble starch 104  7  4 2 1 (100%)(7%)  (4%) (2%) (1%)In this table, each value in the parentheses means a relative percentageto the ethanol amount (100%) just after its preparation.

As evident from the result in Table 2, CTS crystalline anhydrate retainsthe adsorbed ethanol more remarkably and evaporates the adsorbed ethanolmore gradually compared to the branched cyclodextrin, anhydroustrehalose, dextrin, and soluble starch. Therefore, it has only theeffect of adsorbing aromatic substance but also satisfactory retainsaromatic substance. Additionally, 24 hours later, it lost adsorbedethanol due to evaporation similarly as in the other saccharides. Theresult shows that the aroma-retaining material of the present inventionis useful for a sustained-releasing agent because it gradually releasesthe aromatic substance.

EXAMPLE C-3

Preparation of Aroma-Retaining Materials Containing CTS CrystallineAnhydrate

Fifty-five kinds of aroma-retaining materials containing CTS crystallineanhydrate prepared by the method described in Example B-5, and oneselected from the group consisting of methanol, ethanol, 1-propanol,1-butanol, 1-pentanol, 1-octanol, 2-propanol, 2-methyl-1-propanol,2-butanol, 2-methyl-2-propanol, 3-methyl-1-propanol, benzyl alcohol,phenethyl alcohol, 2-aminoethanol, diacetone alcohol, 1,4-butanediol,1-hexanol, O-methoxyphenol, 3-phenyl-1-propanol, benzene, toluene,benzyl chloride, chloroform, ethyl acetate, diethyl ether, petroleumether, α-terpinene, α-pinene, β-pinene, α-terpineol, terpinen-4-ol,limonene, geraniol, linalool, citronellal, citronellol,γ-dodecanolactone, pentylacetate, bornylacetate, allylisothiocyanate,t-butylisothiocyanate, hinokithiol, lemon oil, rosemary oil, lavenderoil, formic acid, acetic acid, propionic acid, butyric acid, valericacid, capronic acid, octanic acid, isobutyric acid, and isovaleric acidwere prepared. The following explains the experimental method in detail.A small amount of each of the above aromatic substances was added to 4 gof CTS in a glass triturator and mixed repeatedly to be adsorbed on CTSuntil the mixture did not keep in a powder form. The amount of theadsorbed aromatic substances was calculated by measuring the weight ofthe resulting powders and subtracting their starting powder weight (4g). The amount of the adsorbed aromatic substances per 1 g of CTScrystalline anhydrate was shown in Table 3. TABLE 3 Amount Amount of ofAdsorbed Adsorbed Substance Substance Aromatic substance (g) Aromaticsubstance (g) Methanol 0.61 α-Pinene 0.35 Ethanol 0.50 β-Pinene 0.301-Propanol 0.46 α-Terpineol 0.33 1-Butanol 0.22 Terpinen-4-ol 0.341-Pentanol 0.31 Limonene 0.42 1-Octanol 0.28 Geraniol 0.32 2-Propanol0.48 Linalool 0.42 2-Methyl-1-Propanol 0.36 Citronellal 0.46 2-Butanol0.29 Citronellol 0.39 2-Methyl-2-Propanol 0.32 γ-Dodecanolactone 0.353-Methyl-1-Propanol 0.36 Pentylacetate 0.46 Benzyl Alcohol 0.43Bornylacetate 0.48 Phenethyl Alcohol 0.40 Allylisothiocyanate 0.372-Aminoethanol 0.13 t-Butylisothiocyanate 0.36 Diacetone Alcohol 0.28Hinokithiol 0.29 1,4-Butanediol 0.28 Lemon Oil 0.28 1-Hexanol 0.41Rosemary Oil 0.31 o-Methoxyphenol 0.42 Lavender Oil 0.293-Phenyl-1-Propanol 0.52 Formic Acid 0.24 Benzene 0.14 Acetic Acid 0.47Toluene 0.29 Propionic Acid 0.50 Benzyl Chloride 0.32 Butyric Acid 0.55Chloroform 0.32 Valeric Acid 0.47 Etyl Acetate 0.41 Capronic Acid 0.47Diethyl Ether 0.17 Octanic Acid 0.47 Petroleum Ether 0.21 IsobutyricAcid 0.51 α-Terpinene 0.37 Isovaleric Acid 0.46

As evident from the result in Table 3, CTS crystalline anhydrate has aneffect of adsorbing aromatic substance in a liquid form such asalcohols, esters, ethers, essential oils, and organic acids, revealingthat it can prepare aroma-retaining materials from various aromaticsubstances in the liquid form.

EXAMPLE C-4

Preparation of Aroma-Retaining Materials Using CTS Crystalline Hydrate,CTS Crystalline Anhydrate, or CTS Amorphous Anhydrate, and Test of TheirProperties

The following example is a test to confirm the effect of CTS crystallinehydrate, CTS crystalline anhydrate, or CTS amorphous anhydrate onretaining, as an aromatic substance, lavender oil, citronellol, orphenethyl alcohol. CTS crystalline pentahydrate prepared by the methoddescribed in Example B-3, CTS crystalline anhydrate in Example B-5, andCTS amorphous anhydrate in Example B-6 were used as the test saccharides“ISOELEAT P®”, a branched cyclodextrin commercialized by MaruhaCorporation, Tokyo, Japan, was used as a control.

In the case of lavender oil, five parts by weight of each of the abovesaccharides were taken into a glass dish sized 90 mm in diameter and 20mm in depth, admixed with one part by weight of lavender oil bystirring. Lavender oil was measured by the method described as follows;admixing 2 ml of diethyl ether with 1 g of each of the above samplesfreshly prepared or after preserved at 25° C. for 10 days; measuring theextracted lavender oil by gas chromatography using “GC-14B” (a gaschromatography commercialized by Shimazu corporation, Kyoto, Japan)equipped with “TC-Wax” (a capillary column sized 0.53 mm in diameter and25 m in length commercialized by GL Sciences Inc., Tokyo, Japan.

In the case of citronellol or phenethyl alcohol, five parts by weight ofeach of the above saccharides were taken into a glass dish sized 90 mmin diameter and 20 mm in depth, admixed with one part by weight ofcitronellol or phenethyl alcohol, and mixed by stirring. The adsorbedcitronellol or phenethyl alcohol was measured by admixing 2 ml ofdiethyl ether with 1 g of each of the above sample freshly prepared orafter preserved at 25° C. for 10 days, and measuring the extractedlavender oil by gas chromatography using “GC-14B” (a gas chromatographydevice commercialized by Shimazu corporation, Kyoto, Japan) equippedwith “DB-5” (a capillary column sized 0.25 mm in diameter and 30 m inlength commercialized by GL Sciences Inc., Tokyo, Japan).

The amount of the retained aromatic substance in each sample wascalculated using the following formula, and the results were shown inTable 4.

Formula:Relative amount of the retained Substance (%)={(an amount of aromaticsubstance in a sample preserved for 10 days)/(an amount of aromaticsubstance in a freshly prepared sample)}×100

TABLE 4 Saccharide Aromatic Amount and Sample Sample Sample substanceRelative Amount 1 2 3 Sample 4 Lavender Amount of the 150 190 185 145Oil Adsorbed Substance (mg/g saccharide) Relative Amount of 1 12 8 0 theRetained Substance (%) Citronellol Amount of the 155 195 190 150Adsorbed Substance (mg/g saccharide) Relative Amount of 30 42 40 9 theRetained Substance (%) Phenethyl Amount of the 140 190 180 130 AlcoholAdsorbed Substance mg/g saccharide) Relative Amount of 3 16 12 0 theRetained Substance (%)Sample 1: CTS Crystalline Hydrate Sample 2: CTS Crystalline AnhydrateSample 3: CTS Amorphous Anhydrate Sample 4: Branched Cyclodextrin

As evident from the result in Table 4, the freshly-prepared samplesretained 150 mg/g of lavender oil in the case of CTS crystallinehydrate, 190 mg/g for CTS crystalline anhydrate, 185 mg/g for CTSamorphous anhydrte, and 145 mg/g for branched cyclodextrin as a control.CTS crystalline anhydrate and CTS amorphous anhydrate had about 1.3-foldhigher adsorbing capacity of branched cyclodextrin. The freshly-preparedsamples contained 155 mg/g of citronellol for CTS crystalline hydrate,195 mg/g for CTS crystalline anhydrate, 190 mg/g for CTS amorphousanhydrate, and 150 mg/g within branched cyclodextrin as a control. CTScrystalline anhydrate and CTS amorphous anhydrate had about 1.2-foldhigher capability of branched cyclodextrin. The freshly-prepared sampleswas contained 140 mg/g of phenethyl alcohol within CTS crystallinehydrate, 190 mg/g within CTS crystalline anhydrate, 180 mg/g within CTSamorphous anhydrate, and 130 mg/g within branched cyclodextrin as acontrol. CTS crystalline anhydrate and CTS amorphous anhydrate had about1.4 to 1.5-fold capability of branched cyclodextrin.

In view of the retaining aromatic substances, in the case of the samplesof lavender oil and phenethyl alcohol, the rate of retaining aromaticsubstance is relatively low. However, in view of the rate of retainedaromatic substance, since CTS in any form, particularly CTS crystallineanhydrate and CTS amorphous anhydrate, have a higher rate than that ofthe branched cyclodextrin as a control, revealing to have a satisfactoryretaining effect. In the case of the samples of citronellol, the rate ofretaining aromatic substance is relatively high. The rate of CTS in anyform is higher than that of the branched cyclodextrin as a control byabout 3.3-fold to 4.7-fold.

Considering the above results, CTS crystalline hydrate, CTS crystallineanhydrate, and CTS amorphous anhydrate have a high capability foradsorbing and retaining aromatic substances depending on the kind ofaromatic substance. Therefore, they are useful as the agent forretaining aroma in the fields of foods, cosmetics, and pharmaceuticals.

EXAMPLE C-5

Herb Extract-Retaining Material

To the mixture of 150 parts by weight of water, 30 parts by weight ofcarrageenan, and 50 parts by weight of hydrolyzed starch were mixed with20 parts by weight of any one of the group consisting of CTS in the formof a pentahydrous crystalline, sucrose (granulated sugar) and “TREHA®”,an α,α-trehalose product commercialized by Hayashibara Shoji, Inc.,Okayama, Japan, and then, the resulting solution was sterilized byheating to 100° C. for 15 min and cooling down to 40° C. One part byweight of a herb extract was admixed with the solution and emulsifiedwith “TK-homomixer”, a mixer commercialized by Tokushu Kika Kogyo Co.,Ltd., Osaka, Japan. By spray-drying the resulting emulsion in aspray-dryer set to 120° C. at the entrance temperature and 80° C. at theexit temperature, a herb-extract-retaining material was obtained. Theamount of the retained aroma substance in the sample was evaluated by 15panels in such a manner of judging the samples about the strength ofaroma after preserved in an opened container at room temperature for twomonths. The result judged in terms of three criteria was shown in Table5. The criterion “good” means strength of aroma when CTS was used as asaccharide. The criterion “slightly good” means that strength of aromais weaker than that with CTS. The criterion “no good” means that aromawas almost lost. TABLE 5 Evaluation Slightly Saccharide Good Good NoGood CTS 15 0 0 Sucrose 0 9 6 α,α-Trehalose 1 13 1

As evidence from the result in Table 5, only one panel of 15 panelsjudged trehalose as the same as in CTS in view of the strength of aroma,however, other panels judged sucrose or α,α-trehalose was interior toCTS as judged “slightly good” or “no good”. The aroma-retaining materialcontaining CTS of the present invention can be advantageously used as anagent for aroma source in the fields of foods, cosmetics, andpharmaceuticals because it is a stable aroma-retaining product duringpreservation. It can be advantageously used for health supplements orraw materials thereof because CTS also functions as a dietary fiber.

EXAMPLE C-6

Grapefruit Aroma-Retaining Material

Five parts by weight of sucrose fatty acid ester HLB15 grade, 45 partsby weight of “TETRUP®”, a syrup commercialized by Hayashibara Shoji,Inc., Okayama, Japan, and 40 parts by weight of CTS crystallinepentahydrate prepared by the method described in Example B-3 wereadmixed and dissolved in 100 parts by weight of water, and then theresulting solution was sterilized by heating at 85 to 90° C. for 15 minand cooling down to about 40° C. The resulting solution was admixed with10 parts weight of grapefruit oil and emulsified. The resulting emulsionwas dried in such a manner of being quickly frozen in −80° C. to loseits moisture, heated up to 50° C., and vacuum-dried for three hours. Thedried resultant was pulverized with a pulverizer to obtain a grapefruitaroma-retaining powder. The product is useful as an agent for aromasource in the fields of foods, cosmetics, pharmaceuticals, andcommodities because it stably retains grapefruit oil.

EXAMPLE C-7

Menthol Aroma-Retaining Material

Twenty parts by weight of CTS amorphous anhydrate prepared by the methoddescribed in Example B-6 and 10 parts by weight of “TREHA®”, ahigh-purity hydrous crystalline a,a-trehalose commercialized byHayashibara Shoji, Inc, Okayama, Japan, were admixed with 10 parts byweight of water, dissolved by heating and cooling down to 50° C. To theresulting solution was admixed one part by weight of menthol to obtain amenthol aroma-retaining material. The product is useful as an agent foraroma source or perfuming agent in the fields of foods, cosmetics,pharmaceuticals, and commodities because it stably retains menthol.

EXAMPLE C-8

Coffee Aroma-Retaining Material

Twenty parts by weight of CTS crystalline pentahydrate prepared by themethod described in Example B-3 and 20 parts by weight of “SUMMALT®”, amaltose commercialized by Hayashibara Shoji, Inc., Okayama, Japan, 10parts by weight of “TETRUP®”, a starch syrup commercialized byHayashibara Shoji, Inc., Okayama, Japan, were admixed with 10 parts byweight of water, dissolved by heating, and cooled down to 50° C. To theresulting solution was added 10 parts by weight of a commercializedcoffee powder to obtain a solution containing coffee. The solution wasdried in such a manner of being quickly frozen in −80° C. to lose itsmoisture, heated up to 60° C., and vacuum-dried for five hours. Thedried resultant was pulverized with a pulverizer to obtain a coffeearoma-retaining powdery product. The product stably retains coffee aromafor a relatively long period of time and can be conveniently used such amanner of being dissolved in a hot water as a coffee beverage havingsatisfactory aroma and sweetness inherent to α,α-trehalose. It can beconveniently used as a raw material for beverage containing coffee andvarious confectioneries containing coffee.

EXAMPLE C-9

Black Tea Aroma-Retaining Material

Fifty parts by weight of CTS crystalline pentahydrate, prepared by themethod described in Example B-3, and 50 parts by weight of “TREHA®”, ahigh-purity hydrous crystalline a,a-trehalose commercialized byHayashibara Shoji, Inc., Okayama, Japan were mixed and dissolved in 50parts by weight of water. The resulting solution was concentrated invacuo to give a concentration of 90% and admixed with five parts byweight of a black tea commercialized by Mitsui Norin Co., Ltd., Tokyo,Japan, and spread on a plastic container, dried by passing through a dryand hot air, and solidified into a block. By pulverizing the resultingblock with a pulverizer and drying the resultant, a powdery product wasobtained. Since the product stably retains black tea aroma for arelatively long period of time, it can be conveniently used in themanner of being dissolved in a hot water and filtrating out thecontained tea leaves to obtain a black tea beverage having asatisfactory aroma and sweetness inherent to α,α-trehalose.

EXAMPLE C-10

Boiled-Dried Fish

Five parts by weight of a syrup, containing CTS and its saccharidederivative(s), prepared by the method described in Example B-1, wasdissolved in 95 parts by weight of boiling water, and further boiled.Ten parts by weight of a raw anchovy were placed in a basket, soaked inthe above solution, boiled, and then, taken out of the basket. Byconventionally drying the boiled anchovy, the captioned product wasobtained. Since the product sufficiently retains its boiled-dried fisharoma, it is useful as a soup stock having a satisfactory flavor.

EXAMPLE C-11

Mirin Powder

Three parts by weight of mirin were mixed with seven parts by weight ofCTS crystalline anhydrate powder prepared by the method described inExample B-6, and the mixture was placed in a container and solidifiedinto a block for two days while converting the CTS into CTS crystallinepentahydrate. By powdering the resulting block with a pulverizer andclassifying, a flavorful aroma-retaining mirin was obtained. The productis useful as a seasoning for instant noodles or clean soups.

EXAMPLE D-1

Juice Powder

Thirty parts by weight of spray-dried grapefruit juice powder, 50 partsby weight of “SUNMALT-S®”, a maltose commercialized by Hayashibara ShojiInc., Okayama, Japan, 10 parts by weight of crystalline anhydrousmaltitol, 10 parts by weight of the grapefruit aroma-retaining materialprepared by the method described in Example C-6, 0.65 part by weight ofanhydrous citric acid, 0.5 part by weight of pullulan, 0.1 part byweight of malic acid, 0.2 part by weight of “AA-2G®”, a2-O-α-glucosyl-L-ascorbic acid commercialized by Hayashibara Shoji,Inc., Okayama, Japan, and 0.1 part by weight of sodium citrate weremixed by stirring and pulverized. The resulting powder was granulated bya fluidized bed granulating machine with a blast temperature of 40° C.,sprayed with an appropriate amount of 70% solution of “SUNMALT-S®”, amaltose commercialized by Hayashibara Shoji, Inc., Okayama, Japan. After30 min of granulation, the captioned product was obtained by weighingand packaging. The product contains about 30% of a juice powder. Theproduct retains grapefruit aroma for a relatively long period of timefree from foreign taste and odor, and it is a high quality juice powder.

EXAMPLE D-2

Chewing Gum

Three parts by weight of a gum base softened by heating and dissolving,two parts by weight of crystalline anhydrous maltitol, two parts byweight of xylitol, two parts by weight of “TREHA®”, a high-purityhydrous crystalline α,α-trehalose commercialized by Hayashibara Shoji,Inc., Okayama, Japan, one part by weight of “SUNMALT-S®”, a maltosecommercialized by Hayashibara Shoji, Inc., Okayama, Japan, 0.5 part byweight of the aroma-retaining material containing menthol prepared bythe method described in Example C-7, appropriate amounts of otheraromatic substances and coloring agents were mixed and kneaded with arolling machine. By shaping and packaging the resultant, the product wasobtained. The product is a flavorful (texture, taste and smell) lightcalorie and anti-dental caries chewing gum retaining menthol aroma for arelatively long period of time.

EXAMPLE D-3

Pullulam Film

Two hundred parts by weight of “PULLULAN PI-20™” a pullulancommercialized by Hayashibara Shoji, Inc., Okayama, Japan, 20 parts byweight of the aroma-retaining material containing menthol prepared bythe method described in Example C-7, 0.1 part by weight of“POLYPHENON™”, a tea-extracted polyphenol commercialized by Mitsui NorinCo., Ltd., Tokyo, Japan, 0.05 part by weight of decanal, and 0.05 partby weight of sun yellow No. 2A were mixed and dissolved in 750 parts byweight of water and then deaerated in vacuo. By spreading the resultingsolution as a film material solution on polyethylene sheet uniformly anddrying by passing through a hot air to obtain a 50° C., pullulan film0.03 mm in thickness. The transparent and glossy film, retaining mentholaroma for a long period of time and having stability against humiditychange, can be used as foods or raw materials for secondary processings.

EXAMPLE D-4

Bath Salt

A bath salt was produced by mixing 90 parts by weight of a roast salt,20 parts by weight of “TREHA®”, a high-purity crystalline hydrousα,α-trehalose by commercialized by Hayashibara Shoji, Inc., Okayama,Japan, one part by weight of anhydrous silicic acid, two parts by weightof “AA-2G”, an ascorbic acid 2-glucoside crystalline powdercommercialized by Hayashibara Shoji, Inc., Okayama, Japan, two parts byweight of the aroma-retaining material containing grapefruit oilprepared by the method described in Example C-6, one part by weight ofaroma-retaining material containing herb extract prepared by the methoddescribed in Example C-5, 0.5 part by weight of “αG HESPERIDIN”, anα-glucosyl hesperidin commercialized by Hayashibara Shoji, Inc.,Okayama, Japan. The product, retaining herb and grapefruit aroma evenafter preservation for a relatively long period of time, is usually usedby diluting with the bath water by 100 to 10,000-folds as a high qualityproduct having the effects of moisturizing, smoothing, and warmingskins.

EXAMPLE D-5

Cosmetic Cream

Two parts by weight of polyoxyethylene glycol monostearate, five partsby weight of glycerin monostearate selfemulsifying, five parts by weightof “TREHA®”, a high purity hydrous crystalline trehalose commercializedby Hayashibara Shoji, Inc., Okayama, Japan, five parts by weight of CTScrystalline pentahydrate prepared by the method described in ExampleB-3, one part by weight of “αG RUTIN”, an α-glucosyl rutincommercialized by Hayashibara Co., Ltd., Okayama, Japan, one part byweight of “AA-2G®”, an ascorbic acid 2-glucoside crystalline powdercommercialized by Hayashibara Shoji, Inc., Okayama, Japan, one part byweight of liquid paraffin, 10 parts by weight of glycerin trioctanoate,and an appropriate amount of an antiseptic were mixed and dissolved byheating. To the resulting solution were added two parts by weight ofL-lactic acid, five parts by weight of 1,3-butylene glycol and 66 partsby weight of purified water and emulsified with a homogenizer. Finally,by mixing the resulting emulsion with an appropriate amount of anaromatic substance, a cosmetic cream was produced. Since the productcontaining CTS retains the aroma and has an antioxidative effect, it isuseful as a high-grade sunburn preventive agent for beauty skin, orwhitening.

EXAMPLE D-6

Shaped Perfume

An incense tree extract was prepared by admixing 100 parts by weight ofincense tree (sandalwood) with 1,000 parts by weight of 70% (w/v)ethanol aqueous solution, standing the mixture at 40° C. for two hours,and filtrating the resultant. Five parts by weight of the above solutionwere diluted by fivefold with the above ethanol aqueous solution. To thesolution was added 60 parts by weight of a “Tabunoki” (Machilusthunbergii) fine powder, 35 parts by weight of a cedar fine powder, sixparts by weight of the CTS crystalline pentahydrate prepared by themethod described in Example B-2, four parts by weight of a freshpreparation of α,α-trehalose as used in Example D-6, and one part byweight of pullulan used in Example D-3, and the resultant was kneadedwith an appropriate amount of water in a usual manner to obtain a paste.The resulting paste was shaped with an oil pressure shaping machine 2 mmin diameter, 136 mm in length, and dried at room temperature (about 10to 20° C.) for three days to obtain a shaped perfume. The product istough and high-grade shaped product because it retains sandalwood aromaand is hardly deteriorated, transformed, and broken.

EXAMPLE E-1

Bactericide

One gram or 2 g of an aroma-retaining material, prepared by mixing CTScrystalline anhydrate and ethanol prepared by the method described inExample C-1, was packaged in a porous paper bag. By packaging andsealing the resulting bag in an aluminium laminate bag, a bactericidewas prepared. The product can be advantageously used as asustained-release bacteriostat and/or bactericide which graduallyreleases ethanol when the contained paper bag is taken off. It is usefulas a bacteriostat and/or bactericide for box lunch and side dishpackaged in a relatively small space because the product only containingnontoxic ethanol and CTS is safe even when administered orally orattached skins or mucosae.

Test for Bactericidal Effect of the Bactericide

A test for bactericidal effect of the aroma-retaining materialconsisting of CTS crystalline anhydrate and ethanol against some typicalresident bacteria, i.e. Bacillus subtilis (ATCC6633), Staphylococcusaureus (ATCC6538), Candida Albicans (ATCC10231), and Aspergillus niger(ATCC16404) was carried out as follow: Bacillus subtilis (ATCC6633) andStaphylococcus aureus (ATCC6538) cultured in “NUTRIENT BROTH”commercialized by Difco Laboratories, USA, at 27° C. for one day, andCandida albicans (ATCC10231) cultered in “YM BROTH” commercialized byDifco Laboratories, USA, at 27° C. for one day were supplied as testsolutions. Aspergillus niger (ATCC16404) cultured in a potato-dextroseagar medium commercialized by Difco Laboratories, USA, at 27° C. forfive days was suspended in the same medium as used in the above cultureand then filtrated. The filtrate containing respectively spores wassupplied as a test solution. 1.5 ml of each test solution was allowed toabsorb unto an adsorbing pad (47 mm in diameter; commercialized byAdvantec Toyo Co., Ltd., Tokyo, Japan), and stood at 25° C. for 30 min.The adsorbing pads containing the bacteria were prepared. An aluminumlaminate bags containing fresh bactericide and bactericide reserved at25° C. for 30 days were opened, and the fresh and reserved bactericidein porous paper bags were taken out. The porous paper bags (containing 1g or 2 g of the bactericide) were placed on glass dishes, and placed ina sealed container (having inner sizes of 30 cm, 21 cm and 9.5 cm and aninner volume of about 5,985 cm³). Then the adsorbing pads containingbacteria were placed in the sealed container at the position of 10 cmapart from each of the paper bags. After incubated at 25° C. for 24hours in the sealed container, viable cells were counted by extractingthe bacteria from the adsorbing pads with physiological saline,appropriately diluting with the saline culturing on nutrient agarplates, and counting the formed colonies. The above results are shownonly about the results of the bactericide freshly-prepared becausepreserved for 30 days one, and there were no difference in thebactericidal effects between the two bactericides. Therefore, theresults with respect to the freshly-prepared bactericide are shown. As acontrol, the same experiment was carried out without ethanol, and theviable cells were counted. The data of viable cells, incubated with thebactericide for 24 hours, are in table 6. TABLE 6 Number of Viable Cells(Number)* After 24 hours bactericide bactericide without (containing(containing bactericide Bacteria Starting 1 g) 2 g) (Control) B.subtilis 1.4 × 10⁴ 1.6 × 10⁴ 0 3.0 × 10⁷ (ATCC 6633) S. aureus 1.4 × 10⁷2.0 × 10⁷ 0 4.1 × 10⁸ (ATCC 6538) C. albicans 2.8 × 10⁷ 2.7 × 10⁷ 0 3.9× 10⁷ (ATCC 10231) A. niger 2.5 × 10³ 2.9 × 10³ 0 3.6 × 10³ (ATCC 16404)NOTE*The number of viable cells per one adsorbing pad absorbing 1.5 ml oftest solution.

As evident from the result in table 6, the bactericide, containing CTSin the form of a crystalline anhydrous, being packaged in the porouspaper bag, and retaining ethanol as a bactericide, released ethanol andexerted bacteriostatic and/or bactericidal effect in a dose dependentmanner. The bacteriostatic effect was revealed in the test using thepaper bags containing 1 g of the bactericide, which tended to inhibitthe increasing number of bacteria. The bactericidal effect was revealedin the paper bags containing 2 g of the bactericide, which completelykilled the bacteria and/or the spore in the container having about 6 Lvolume within 24 hours. The above result shows that the bactericide ofthe present invention has a satisfactory bacteriostatic and/orbacteriocidal effect.

Industrial Applicability

As described above, according to the present invention, thearoma-retaining material having the effect of adsorbing and retainingaroma and being satisfactory stable can be obtained without affectingaroma or aroma-retaining material by mixing an aromatic substance andCTS or one or more members selected from the group consisting of syrup,mascuites, solid, and powder containing CTS and its saccharidederivative(s). It also enables to produce an aroma-retaining compositionmore easily. Since the aroma-retaining material of the present inventionhas retained-release property, it is not useful as onlysustained-releasing agent for retaining aroma but alsosustained-releasing bacteriostat and/or bactericide in case of thearomatic substance having a bactericidal effect. Thus, the presentinvention will give a great use on the industrial fields, for example,it provides a product having a satisfactory sustained aroma andstability.

“Bacillus globisporus C9” (FERM BP-7143), described in thisspecification, was deposited and accepted on Apr. 25, 2000, inInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology, Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan.

1. A method for retaining aroma characterized in that it comprises astep of incorporating a cyclic tetrasaccharide represented by ChemicalFormula 1 or a mixture of the cycle tetrasaccharide and its saccharidederivative into an aromatic substance or a composition comprising saidaromatic substance: Chemical formula 1:


2. The method of claim 1, characterized in that wherein said aromaticsubstance is a natural aromatic substance derived from an animal orplant, a composition comprising said natural aromatic substance, or asynthetic aromatic substance.
 3. The method of claim 1 or 2,characterized in that wherein said aromatic substance is ethanol and/oracetic acid.
 4. The method of claim 1, 2, or 3, characterized in thatwherein said cyclic tetrasaccharide or said mixture is added in anamount of 1 to 10,000 parts by weight to one part by weight of saidaromatic substance on a dry solid basis of said cyclic tetrasaccharide.5. The method of any one of claims 1 to 4, characterized in that whereinsaid cyclic tetrasaccharide or said mixture is in the form of a syrup,mascuite, solid, amorphous powder, hydrous crystalline powder, ofanhydrous crystalline powder.
 6. The method of any one of claims 1 to 5,characterized in that it further comprises a step of incorporating oneor more members selected from the group consisting of monosaccharides,oligosaccharides, and polysaccharides with said cyclic tetrasaccharideor said mixture.
 7. The method of any one of claims 1 to 6,characterized in that wherein said cyclic tetrasaccharide or saidmixture is incorporated into said composition in the presence of waterand/or an emulsifier.
 8. The method of any one of claims 1 to 7,characterized in that wherein said cyclic tetrasaccharide or saidmixture is produced from starch or phytoglycogen.
 9. An aroma-retainingmaterial, which is obtainable by any one of the methods of claims 1 to8.
 10. The aroma-retaining material of claim 9, characterized in thatwherein said aromatic substance is in a composition comprising any oneof alcohols, seasonings, and fermented foods.
 11. The aroma-retainingmaterial of claim 9, characterized in that wherein said aromaticsubstance is ethanol and/or acetic acid.
 12. The aroma-retainingmaterial of claim 9, 10 or 11, characterized in that it is in the formof a liquid, semisolid, solid, or powder.
 13. The aroma-retainingmaterial of any one of claims 9 to 12, characterized in that whereinsaid aromatic substance has a sustained-release property.
 14. Thearoma-retaining material of any one of claims 9 to 13, characterized inthat it has a bacteriostat or bactericide effect.
 15. A composition,which is prepared by co-existing or incorporating any one of saidaroma-retaining materials of claims 9 to
 14. 16. The composition ofclaim 15, characterized in that it is in the form of a food, cosmetic,pharmaceutical, or commodity.
 17. An aroma-retaining agent, whichcomprises a cyclic tetrasaccharide represented by Chemical Formula 1 ora mixture of said cyclic tetrasaccharide and its saccharide derivativeas effective ingredients.
 18. A sustained-releasing agent having anaromatic substance, which comprises a cyclic tetrasaccharide representedby Chemical formula 1 or a mixture of said cyclic tetrasaccharide andits saccharide derivative.
 19. A bacteriostat and/or bactericide,characterized in that it comprises ethanol and/or acetic acid, and acyclic tetrasaccharide or a mixture of said cyclic tetrasaccharide andits saccharide derivative.
 20. The bacteriostat and/or bactericide ofclaim 19, characterized in that it gradually releases ethanol and/oracetic acid.
 21. A method for bacteriostasis and/or sterilization offoods, cosmetics, pharmaceuticals, or commodities, characterized in thatsaid bacteriostat and/or bactericide of claim 19 or 20 are co-existedand/or incorporated into said foods, cosmetics, pharmaceuticals, orcommodities.